N-Cyclopropyl-(20R)-2-Methylene-19,26,27-Trinor-25-Aza-Vitamin D Analogs and Their Uses

ABSTRACT

This invention discloses N-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-vitamin D analogs, and specifically N-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitamin D 3  and pharmaceutical uses therefor. This compound exhibits relatively high binding activity and pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent especially for the treatment or prevention of leukemia, colon cancer, breast cancer, skin cancer or prostate cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/487,057, filed May 17, 2011, which is incorporated by referenceherein in its entirety for any purpose.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly toN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-vitamin D analogsand their pharmaceutical uses, and especiallyN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃, its biological activities, and its pharmaceutical uses.

The most active metabolite of vitamin D₃, namely, 1α,25-dihydroxyvitaminD₃, is a potent calcium and phosphorous regulating hormone playing animportant role in bone homeostasis in animals and humans. Also, inaddition to this classical role, the natural hormone elicitsimmunomodulation as well as cell differentiation and proliferationactivities in numerous malignant cells and keratinocytes [Feldman et al,Vitamin D, 2^(nd) ed,; Elsevier Academic Press: New York, 2005].1α,25-Dihydroxyvitamin D₃ expresses these functions by binding to thevitamin D receptor (VDR), a ligand-regulated transcription factor.Structural analogs of this metabolite have been prepared and tested suchas 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, and various otherside-chain and A-ring modified vitamins. Some potent synthetic analogshave been used clinically to treat bone disorders such as osteoporosisand the skin disorder psoriasis. Some of these compounds exhibitseparation of activities in cell differentiation and calcium regulation.The difference in activity may be advantageous in treating a variety ofdiseases such as renal osteodystrophy, vitamin D-resistant rickets,osteoporosis, and malignancies.

In U.S. Pat. Nos. 4,800,198, 5,089,641 and 5,366,731, a class ofsecosterol compounds has also been prepared which exhibit high growthinhibitory activity towards malignant cells, such as leukemia cells, buthave significantly less of the undesired side-effects (potent calcemicaction) of some of the known vitamin D compounds mentioned above. Thisselectivity and specificity of action makes the secosterols potentiallyuseful as agents for the treatment of malignancies such as leukemia.These secosterol compounds have also been proposed for use indermatological compositions to treat skin disorders such as dermatitis,eczema and psoriasis, as well as in cosmetic compositions to treat lesssever skin conditions such as wrinkles, lack of dermal hydration, i.e.dry skin, lack of adequate skin firmness, i.e. skin slackness, andinsufficient sebum secretion.

Calcipotriene is another synthetic vitamin D₃ derivative that ismarketed for topical dermatological use against psoriasis. Calcipotrienehas a 24-cyclopropyl substituent, and is disclosed in U.S. Pat. No.4,866,048.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of 19-nor-analogs such as1α,25-dihydroxy-19-nor-vitamin D₃ revealed a selective activity profilewith high potency in inducing cellular differentiation, and reducedcalcium mobilizing activity. Thus, these compounds may be potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman. et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, ED-120, and fluoroalkyl groups)A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also been prepared andtested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111 (1993) Nishii etal., Osteoporosis Int. Suppl. 1, 190(1993); Posner et al., J. Org. Chem,59, 7855 (1994), and J. Org. Chem, 60, 4617 (1995)).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

SUMMARY OF THE INVENTION

The present invention is directed towardN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-vitamin Danalogs, and their pharmaceutical uses, and more specifically towardN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃, its biological activity, and various pharmaceutical uses for thiscompound.

Structurally theseN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-vitamin D analogsare characterized by the general formula I shown below:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from the group consisting of hydrogen and a hydroxy-protectinggroup.

The preferred analog isN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃ (referred to herein as “CPA- 1”) which has the following formula Ia:

The above compounds of formula I, especially formula Ia, exhibit adesired, and highly advantageous, pattern of biological activity. Thesecompounds are characterized by relatively high binding to vitamin Dreceptors, i.e. they bind with only slightly lower affinity than thenatural hormone 1α,25-dihydroxyvitamin D₃ They are equally as potent incausing differentiation of HL-60 cells as 1,25(OH)₂D₃. They also exhibitrelatively low transcriptional activity as well as relatively lowactivity in their ability to mobilize calcium from bone, but are quitepotent in their ability to promote intestinal calcium transport, ascompared to 1α,25-dihydroxyvitamin D₃.

The above compounds I, and particularly Ia, have relatively high bindingaffinity, are characterized by some cell-type selectivity and causedifferentiation of cancer cells nearly as well as the native hormone buthave notably lower potency in raising tissue calcium levels. Thus, thesecompounds, especially CPA-1, have potential as anti-cancer agents andprovide therapeutic agents for the prevention or treatment of leukemia,colon cancer, breast cancer, skin cancer and prostate cancer.

One or more of the compounds may be present in a composition to treat orprevent the above-noted diseases in an amount from about 0.01 μg/gm toabout 1000 μg/gm of the composition, preferably from about 0.1 μg/gm toabout 500 μg/gm of the composition, and may he administered topically,transdermally, orally, rectally, nasally, sublingually, or parenterallyin dosages of from about 0.01 μg/day to about 1000 μg/day, preferablyfrom about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 illustrate various biological activities ofN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃, hereinafter referred to as “CPA-1,” as compared to the nativehormone 1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of CPA-1 and1,25(OH)-₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of CPA-1 and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to CPA-1;

FIG. 4 is a bar graph illustrating the bone calcium mobilizationactivity of 1,25(OH)₂D₃ as compared to CPA-1; and

FIG. 5 is a bar graph illustrating the intestinal calcium transportactivity of 1,25(OH)₂D₃ as compared to CPA-1.

DETAILED DESCRIPTION OF THE INVENTION

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkl-O—CO— groupings such asmethoxycarhonyl, ethoxycarbonyl, propoxycarbonyl, isopropxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, in all of its isomeric forms,or a carboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl,malonyl, succinyl, glutaryl group, or an aromatic acyl group such asbenzoyl, or a halo, nitro, or alkyl substituted benzoyl group. The word“alkyl” as used in the description or the claims, denotes astraight-chain or branched alkyl radical of 1 to 10 carbons, in all itsisomeric forms. “Alkoxy” refers to any alkyl radical which is attachedby oxygen, i.e. a group represented by “alkyl-O—.” Alkoxyalkylprotecting groups are groupings such as methoxymethyl, ethoxymethyl,methoxyethoxymethyl, or tetrahydrafuranyl and tetrahydropyranyl.Preferred silyl-protecting groups are trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, dibutylmethylsilyl, diphenylmethylsilyl,phenyldimethylsilyl, diphenyl-t-butylsilyl and analogous alkylated silylradicals. The term “aryl” specifies a phenyl-, or an alkyl-, nitro- orhalo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,deuteroalkyl” and “fluoroalkyl” refers to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively. An“alkylidene” refers to a radical having the general formula C_(k)H_(2k)—where k is an integer.

N-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃ (referred to herein as “CPA-1”), a 19-nor vitamin D analog which ischaracterized by the presence of a methylene substituent at the carbon 2(C-2), and the methyl groups normally located at the 26 and 27 positions(C-26 and C-27) as well as the hydroxyl substituent normally attached tothe 25-position (C-25) in the side chain, replaced with an aza grouplocated at carbon atom 25 (C-25) in the side chain, was synthesized andtested. Such vitamin D analog seemed an interesting target because therelatively small methylene group at the C-2 position should notinterfere with binding to the vitamin D receptor. Structurally, this19-nor analog is characterized by the general formula Ia previouslyillustrated herein, and its pro-drug (in protected hydroxy form) ischaracterized by general formula I previously illustrated herein.

The preparation ofN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-vitamin D analogshaving the structure I can be accomplished by a common general method,i.e. the condensation of a bicyclic Windaus-Gundmann type ketone II withthe allylic phosphine oxide III to the corresponding2-methylene-19-nor-vitamin D analog I followed by deprotection atpositions C-1, C-3 and C-25 in the latter compound (see Schemes I and IIherein):

In the structures I, II and III, groups X₁, X₂ and X₃ arehydroxy-protecting groups, preferably X₁ and X₂ are t-butyldimethylsilyl(TBS), and X₃ is tert-butyloxycarbonyl (BOC), it being also understoodthat any functionalities that might be sensitive, or that interfere withthe condensation reaction, be suitably protected as is well-known in theart. The process shown above represents an application of the convergentsynthesis concept, which has been applied effectively for thepreparation of vitamin D compounds [e.g. Lythgoe et al., J. Chem. Soc.Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Tohet al., J. Org. Chem. 48 1414 (1983); Baggiolini et al., J. Org. Chem,51, 3098 (1986); Sardina et al., J. Org. Chem. 51, 1264 (1986); J. Org.Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca etal., U.S. Pat. No. 5,536,713.

The hydrindanone of the general structure II is not known. It can beprepared by the method shown in Schemes I and II herein (see thepreparation of compound CPA-1).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compounds I and Ia isillustrated and described more completely in U.S. Pat. No. 5,843,928entitled “2-Alkylidene-19-Nor-Vitamin D Compounds” the specification ofwhich is specifically incorporated herein by reference.

More specifically, reference should be made to the followingillustrative example and description as well as to Schemes I and IIherein for a detailed illustration of the preparation of compound CPA-1.

This invention is described by the following illustrative examples. Inthese examples specific products identified by Arabic numerals (e.g., 1,2, 3, etc.) refer to the specific structures so identified in thepreceding description and in the Scheme I and Scheme II.

EXAMPLES

Chemistry. Melting points (uncorrected) were determined on aThomas-Hoover capillary melting-point apparatus. Optical rotations weremeasured in chloroform using a Perkin-Elmer 241 automatic polarimeter at22° C. Ultraviolet (UV) absorption spectra were recorded with aPerkin-Elmer Lambda 3B UV-VIS spectrophotometer in ethanol. ¹H nuclearmagnetic resonance (NMR) spectra were recorded in deuteriochloroform at200, 400 and 500 MHz with a Varian Unity, Bruker DMX-400 and BrukerDMX-500 spectrometers, respectively. ¹³C nuclear magnetic resonance(NMR) spectra were recorded at 50, 100 and 125 MHz with the samespectrometers in deuteriochloroform. Chemical shifts (δ) were reporteddownfield from internal Me₄Si (δ 0.00). Electron impact (EI) massspectra were obtained with a Micromass AutoSpec (Beverly, Mass.)instrument. High-performance liquid chromatography (HPLC) was performedon a Waters Associates liquid chromatograph equipped with a Model 6000Asolvent delivery system, a Model U6K Universal injector, and a Model 486tunable absorbance detector. THF was freshly distilled before use fromsodium benzophenone ketyl under argon.

Example I Preparation of (8S,20S)-de-A,B-20-(hydroxymethyl)pregnan-8-ol(1)

Ozone was passed through a solution of vitamin D₂ (3 g, 7.6 mmol) inmethanol (250 mL) and pyridine (2.44 g, 2.5 mL, 31 mmol) for 50 min at−78° C. The reaction mixture was then flushed with an oxygen for 15 minto remove the residual ozone and the solution was treated with NaBH₄(0.75 g, 20 mmol). After 20 min the second portion of NaBH₄ (0.75 g, 20mmol) was added and the mixture was allowed to warm to room temperature.The third portion of NaBH₄ (0.75 g, 20 mmol) was then added and thereaction mixture was stirred for 18 h. The reaction was quenched withwater (40 mL) and the solution was concentrated under reduced pressure.The residue was extracted with ethyl acetate and the combined organicphases were washed with 1M aq. HCl, saturated aq. NaHCO₃, dried (Na₂SO₄)and concentrated under reduced pressure. The residue was chromatographedon silica gel with hexane/ethyl acetate (75:25) to give the diol 1 (1.21g, 75% yield) as white crystals:

m.p. 106-108° C.; [α]_(D)+30.2° (c 1.46, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ 408 (1H, d, J=20 Hz, 8α-H), 3.63 (1H, dd, J=10.5, 3.1 Hz, 22-H), 338(1H, dd, J=10.5, 6.8 Hz, 22-H), 1.99 (1H, br.d, J=13.2 Hz), 1.03 (3H, d,J=6.6 Hz, 21-H₃), 0.956 (3H, s, 18-H₃); ¹³C NMR (100 MHz) δ 69.16 (d,C-8), 67.74 (t, C-22), 52.90 (d), 52.33 (d), 41.83 (s, C-13), 40.19 (t),38.20 (d), 33.53 (t), 26.62 (t), 22.54 (t), 17.36 (t), 16.59 (q, C-21),13.54 (q, C-18); MS (EI) m/z 212 (2, M⁺), 194 (34, M⁺ —H₂O), 179 (33, M⁺—H₂O—CH₃), 163 (18, M⁺ —CH₂OH—H₂O), 135 (36), 125 (54), 111 (100), 95(63), 81 (67); exact mass calculated for C₁₃H₂₂O (M⁺ —H₂O) 194.1671,found 194.1665.

Preparation of(8S,20S)-de-A,B-8-triethylsilyloxy-20-(acetyloxymethyl)pregnane (2)

Acetic anidryde (0.41 g, 0.40 mL, 4.0 mmol) was added to a solution ofthe diol 1 (0.5 g, 2.3 mmol) and Et₃N (1.64 mL, 11.7 mmol) in anhydrousCH₂Cl₂ (20 mL) at room temperature (rt). The reaction mixture wasstirred at rt for 24 h, diluted with methylene chloride (100 mL), washedwith 5% aq. HCl, water, saturated aq. NaHCO₃, dried (Na₂SO₄) andconcentrated under reduced pressure. The residue (0.68 g) waschromatographed on silica gel with hexane/ethyl acetate (75:25) to givethe desired alcohol (0.53 g, 88% yield) as a colorless oil.

To a stirred solution of the alcohol (0.53 g, 2.1 mmol) and 2,6-lutidine(0.29 mL, 0.26 g, 2.5 mmol) in anhydrous methylene chloride (5 mL)triethylsilyl trifluoromethane-sulfonate (0.54 mL, 2.5 mmol) was addedat 0° C. The reaction mixture was allowed to warm to room temperature (1h), and stirring was continued for additional 30 min. Methylene chloridewas added and the mixture was washed with water, dried (Na₂SO₄) andconcentrated under reduced pressure. The residue was chromatographed onsilica gel with hexane/ethyl acetate (97:3) to afford the product 2(0.74 g, 95% yield) as a color less oil:

[α]_(D)+40.77 (c 4.9, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.06 (2H, m),3.77 (1H, dd, J=10.64, 3.08 Hz, 22-H), 2.05 (3H, s), 1.93 (1H, br. d,J=12.4 Hz), 0.98 (3H, d, J=6.6 Hz, 21-H₃), 0.96 (9H, t, J=7.9 Hz), 0.92(3H, s), 0.56 (6H, q, J=7.9 Hz); ¹³C NMR (100 MHz) δ 171.4, 69.61,69.25, 53.44, 52.85, 42.26, 40.64, 35.39, 34.62, 26.80, 23.07, 21.03,17.67, 17.09, 13.57, 6.95, 4.96; exact mass calculated for C₁₉H₃₅O₃Si (M—C2H5) 339.2355, found 339.2347.

Preparation of(8S,20S)-de-A,B-8-triethylsityloxy-20-(hydroxymethyl)pregnane (3).

The acetate 2 (0.58 g, 1.6 mmol) was treated with a solution of NaOH (1g, 25 mmol) in anhydrous ethanol (20 mL) at room temperature. Afterstirring of the reaction mixture for 3 h, ice and 5% aq. HCl were addeduntil pH=6. The solution was extracted with ethyl acetate (3×50 mL) andthe combined organic phases were washed with saturated aq. NaHCO₃, dried(Na₂SO₄) and concentrated under reduced pressure. The residue waschromatographed on silica gel with hexane/ethyl acetate (75:25) to givethe alcohol 3 (0.44 g, 84% yield) as a colorless oil.

[α]_(D)+41.1 (c 2.85, CHCl₃); ¹H NMR (400 MHz, CDCl₃+TMS) δ 4.04 (1H, d,J=2.4 Hz, 8α-H), 3.63 (1H, dd, J=10.5, 3.2 Hz, 22-H), 3.38 (1H, dd,J=10.5, 6.8 Hz, 22-H), 1.94 (1H, br.d, J=12.4 Hz), 1.02 (3H, d, J=6.6Hz, 21-H₃), 0.95 (9H, t, J=7.9 Hz), 0.92 (3H, s, 18-H₃), 0.55 (6H, q,J=7.9 Hz); ¹³C NMR (100 MHz) δ 69.24, 67.93, 53.07, 52.84, 42.11, 40.61,38.27, 34.58, 26.79, 23,03, 17.63, 16.64, 13.55, 6.91, 4.90; exact masscalculated for C₁₉H₃₈O₂Si (M⁺) 326.2641, found 326.2626.

Preparation of (8S,20S)-de-A,B-8-triethylsiloxy-20-formylpregnane (4)

To a solution of DMSO (1.2 mL) in CH₂Cl₂ (2 mL) at −60° C. oxalylchloride (1.11 g, 8.74 mmol) was added. After 2 min, a solution of theprimary alcohol 3 (0.22 g, 0.67 mmol) in anhydrous CH₂Cl₂ (2 mL) at −60°C. was added via cannula. The resulting mixture was stirred at −60° C.for 2 h, quenched with Et₃N (4.9 mL), and warmed up to room temperature.Upon dilution with H₂O, the mixture was extracted with CH₂Cl₂, dried(MgSO₄), filtered, concentrated, and purified by flash columnchromatography (9.5:0.5 Hexane/EtoAc; Rf=0.12) to give the desiredaldehyde 4 (0.120 mg, 0.37 mmol, 54% yield) as an oil.

¹H NMR (200 MHz, CDCl₃) δ 9.57 (1H, d, J=3.0 Hz, CHO), 4.06 (1H, d,J=2.4 Hz, 8α-H), 2.38 (1H, m, 20-H), 1.09 (3H, d, J=6.8 Hz, 21-H₃), 0.95(12H, m, Si(CH₂CH₃)₃+18-H₃), 0.55 (6H, q, J=7.8 Hz, Si(CH₂CH₃)₃).

Preparation of(8S,20S)-de-A,B-8-triethyisilyloxy-20-[2-(methoxycarbonyl)-et-(1E)-en-yl]pregnane(5)

To a solution of the aldehyde 4 (0.120 g, 0.37 mmol) in absolute EtOH (3mL) at 0° C. was added methyl(triphenylphosphoranylidene)-acetate (0.307g, 0.92 mmol) and Et₃N (0.037 g, 0.37 mmol). The mixture was stirred atr.t. for 24 h and then the solvent was evaporated. The residue waspurified by flash column chromatography (9.5:0.5 Hexane/EtOAc, Rf=0.45)to obtain 5 (0.105 g, 0.27 mmol, 75% yield) as an oil.

[α]_(D)+58.5 (c 2.37, CH₂Cl₂) ¹H NMR (400 MHz, CDCl₃±TMS) δ 6.83 (1H,dd, J=15.06, 8.96, Hz), 5.73 (1H, d, J=15.6 Hz), 4.03 (1H, d, J=2.4 Hz,8α-H), 3.76 (3H, s) 1.94 (1H, br.d, J=12.4 Hz), 1.05 (3H, d, J=6.6 Hz,21-H₃), 0.95 (12H, m), 0.55 (6H, q, J=7.9 Hz); ¹³C NMR (100 MHz) δ167.56, 155.28, 118.41, 69.22, 55.54, 52.89, 51.35, 42.44, 40.63, 39.41,34.56, 27.32, 22.96, 19.08, 17.64, 13.76, 6.94, 4.91; exact masscalculated for C₂₀H₃₆O₃Si (M⁺-Et) 351.2350, found 351.2366.

Preparation of(8S,20R)-de-A,B-8-triethylsilyloxy-20-[2-(methoxycarbonyl)ethyl]pregnane(6)

A solution of the compound 5 (0.105 g, 0.27 mmol) in absolute EtOH (5mL) was hydrogenated for 9 h in the presence of 10% palladium onpowdered charcoal (15 mg). The reaction mixture was filtered through abed of Celite with several ethanol washes, the filtrate was concentratedand the residue was chromatographed on silica gel with Hexane/EtOAc(97:3, Rf=0.47) to give the product 6 (0.092 g, 0.24 mmol, 87% yield).¹H NMR (200 MHz, CDCl₃) δ 4.02 (1H, broad signal, 8α-H), 3.60 (2H, t,J=6.2 Hz, 24-H₂), 0.94 (15H, m, Si(CH₂CH₃)₃+18-H₃+21-H₃), 0.54 (6H, q,J=7.14 Hz, Si(CH₂CH₃)₃).

Preparation of(8S,2R)-de-A,B-8-triethylsilyloxy-20-(hydroxypropyl)pregnane (7)

A solution of the compound 6 (0.092 g, 0.24 mmol) in THF (1 mL), wasadded to a solution of LiAlH₄ 1M in THF (0.48 mL, 0.48 mmol) cooled at−10° C. The reaction mixture was stirred at r.t. for 3 h, then water(0.2 mL) and NaOH 1M (0.05 mL) were added, and the resulting suspensionwas filtered off. The evaporation of the solvent afforded the alcohol 7(0.070 g, 0.24 mmol, 99% yield) as a clear oil.

¹H NMR (400 MHz, CDCl₃+TMS) δ 4.03 (1H, d, J=2.4 Hz, 8α-H), 3.62 (2H, m)1.96 (1H, br.d, J=12.4 Hz), 0.95 (15H, m), 0.55 (6H, q, J=7.9 Hz); ¹³CNMR (100 MHz) δ, 69.40, 63.66, 56.69 53.10, 42.13, 40.79, 35.08, 34.65,31.68, 29.69, 29,41, 27.31, 23.00, 18.60, 17.69, 13.52, 6.95, 4.95;exact mass calculated for C₂₁H₄₂O₂Si (M⁺) 354.2949, found 354.2943

Preparation of(8S,20R)-de-A,B-8-triethylsityloxy-20-[3-(cyclopropylamine)propyl]pregnane(9)

To a solution of DMSO (1.2 mL) in CH₂Cl₂ (2 mL) at −60° C. oxalylchloride (0.34 g, 2.91 mmol) was added. After 2 min, a solution of theprimary alcohol 7 (0.081 g, 0.28 mmol) in anhydrous CH₂Cl₂ (2 mL) at−60° C. was added via cannula. The resulting mixture was stirred at −60°C. for 2 h, quenched with Et₃N (4.9 mL), and warmed up to roomtemperature. Upon dilution with H₂O, the mixture was extracted withCH₂Cl₂, dried (MgSO₄), filtered, concentrated, and purified by flashcolumn chromatography (9.5:0.5 Hexane/EtOAc; Rf=0.55) to give thedesired aldehyde 8 (0.046 mg, 0.16 mmol, 57% yield) which wasimmediately used for the next step.

¹H NMR (200 MHz, CDCl₃) δ 9.76 (1H, s, CHO), 4.02 (1H, br signal, 8α-H),2.40 (2H, m, 23-H₂), 0.94 (15H, m, Si(CH₂CH₃)₃+18-H₃+21-H₃), 0.54 (6H,q, J=8.05 Hz, Si(CH₂CH₃)₃).

To a solution of the aldehyde 8 (0.046 g, 0.16 mmol) in anhydrous CH₂Cl₂(1 mL) was added cyclopropylamine (0.0092 g, 0.16 mmol) and the mixturewas cooled at 0° C. before adding sodium triacethoxyborohydride (0.047g, 0.22 mmol). The reaction mixture was stirred at r.t. for 2 h. Thensat. aq. NaHCO₃ solution (1 mL) was added and the mixture was extractedwith EtOAc. The organic phase was dried (MgSO₄), filtered, and thesolvent was evaporated to give 9 (0.049 g, 0.15 mmol, 93% yield).

[α]_(D)+44.5 (c 1.12, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃+TMS) δ 4.02 (1H,d, J=2.4 Hz, 8α-H), 2.65 (2H, m), 2.13 (1H, m), 0.95 (9H, t, J=7.9 Hz),0.89 (6H, m), 0.55 (6H, q, J=7.9 Hz), 0.43 (2H, m), 0.36 (2H, m); ¹³CNMR (100 MHz) δ, 69.40, 56.70, 53.09, 50.21, 42.11, 40.78, 35.20, 34.65,33.40, 30.35, 27.33, 26.45, 23.01 18.61, 17.69, 13.51, 6.95, 6.16, 6.13,4.94; exact mass calculated for C₂₄H₄₈NOSi (MH)⁺ 394.3505, found394.3506.

Preparation of(8S,20R)-de-A,B-8-triethylsilyloxy-20-[3-(cyclopropyl-N-t-Boc-amine)propyl]pregnane(10)

To a solution of compound 9 (0.033 g, 0.084 mmol) in CH₃CN (3 mL) Boc₂O(0.022 g, 0.10 mmol) and DMAP (0.001 g, 0.0084 mmol) were added undervigorous stirring. After stirring at r.t. for 1 h, the mixture wasdiluted with EtOAc, washed with water and brine then dried over MgSO₄.Concentration gave the desired compound 10 (0.040 g, 0.081 mmol, 96%yield). ¹H NMR (400 MHz, CDCl₃+TMS) δ 4.02 (1H, d, J=2.4 Hz, 8α-H), 3.14(2H, m), 2.49 (1H, m), 1.95 (1H, br.d, J=124 Hz), 1.46 (9H, s), 0.94(9H, t, J=7.9 Hz), 0.89 (6H, m), 0.73 (2H, m), 0.56 (8H, m).

Preparation of(8S,20R)-de-A,B-20-[3-(cyclopropyl-N-t-Boc-amine)propyl]pregnan-8-one(12)

The protected alcohol 10 (0.032 g, 0.065mmol) was dissolved in anhydrousTHF (5 mL) and TBAF 1M in THF (130 ρL, 0.13 mmol) was added. After 5 hof stirring at r.t. the reaction mixture was diluted with EtOAc, washedwith brine, dried (MgSO₄) and concentrated under reduced pressure. Theresidue was purified by flash column chromatography on silica gel withHexane/EtOAc (9.5:0.5) to give the alcohol 11 (0.016 g, 0.042 mmol, 65%yield). ¹H NMR (400 MHz, CDCl₃+TMS) δ 4.07 (1H, s, 8α-H), 3.14 (2H, m),2.48 (1H, m), 1.98 (1H, br.d, J=12.4 Hz), 1.46 (9H, s), 0.93 (3H, s),0.91 (6H, m), 0.73 (2H, m), 0.57 (2H, m).

Pyridinium dichromate (0.079 g, 0.211 mmol) was added to a solution ofthe alcohol 11 (0.016 g, 0.042 mmol) and pyridinium p-toluenesulfonate(0.007 g, 0.030 mmol) in anhydrous CH₂Cl₂(5 ml). The resultingsuspension was stirred at r.t. for 3 h. The reaction mixture wasfiltered through a Waters silica Sep-pack cartridge (5 g) that wasfurther washed with Hexane/EtOAc (9.5:0.5). After removal of the solventthe ketone 12 (0.014 g, 0.037 mmol, 88% yield) was recovered as acolorless oil. ¹H NMR (400 MHz, CDCl₃+TMS) δ 3.15 (2H, m), 2.45 (2H, m),1.46 (3H, s), 0.96 (3H, d, J=5.7 Hz), 0.73 (2H, m), 0.64 (3H, s), 0.58(2H, m); ¹³C NMR (100 MHz) δ, 212.08, 156.76, 79.18, 61.94, 56.59,49.89, 40.95, 38.95, 35.26, 32.79, 28.47, 27.47, 24.85, 24.04, 19.0418.69, 12.48, 7.99, exact mass calculated for C₂₃H₃₉NO₃Na(MNa)⁺400.2823, found 400.2821.

Preparation ofN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃ (15)

To a solution of phosphine oxide 13 (0.086 g, 0.148 mmol) in anhydrousTHF (0.5 mL) at −20° C. was slowly added PhLi (1.7 M in di-n-butylether,0.087 mL, 0.148 mmol) under argon with stirring. The solution turneddeep orange. After 30 min the mixture was cooled at −78° C. and aprecooled (−78° C.) solution of ketone 12 (0.014 g, 0.037 mmol) in atleast 200 μL of anhydrous THF was slowly added. The mixture was stirredunder argon at −78° C. for 3 h and at 0° C. for 18 h. Ethyl acetate wasadded, and the organic phase was washed with brine, dried (MgSO₄) andevaporated. The residue was dissolved in hexane and applied on a Waterssilica Sep-Pack cartridge (2 g). The cartridge was washed with hexaneand hexane/ethyl acetate (99.5:0.5) to give the 19-norvitamin 14 (0.021g, 0.028 mmol, 76% yield).

¹H NMR (CDCl₃, 900 MHz) δ 6.20 (1H, d, J=10.8 Hz), 5.82 (1H, d, J=10.8Hz,), 4.96 (1H, s,), 4.91 (1H, s,), 4.41 (2H, m). 3.15 (2H, m), 2.81(1H, dm, J=12.6 Hz), 2.52 (1H dd, J=13.5, 6.3 Hz). 2.45 (1H, dd, J=12.6,4.5 Hz), 2.32 (1H, dd, J=13.5, 2.7 Hz), 2.17 (1H, dd, J=12.6, 8.1. Hz),1.46 (9H, s), 0.94 (3H, d, J=7.2 Hz), 0.89 (9H, s), 0.86 (9H, s,), 0.73(2H, m), 0.58 (2H, m), 0.54 (3H, s) 0.098 (3H, s) 0.096 (3H, s), 0.073(3H, s), 0.055 (3H, s); ¹³C NMR (CDCl₃, 200.9 MHz) δ 157.03, 153.20,141.39, 132.99, 122.63, 116.36, 106.48, 72.75, 71.79, 56.72, 56.48,47.84, 45.89, 40.81, 38.78, 36.14, 33.20, 28.96, 28.73, 27.92, 26.06,23.65, 22.43, 19.04, 18.48, 18.39, 12.32, 8.49- 4.63, −4.88; exact masscalculated for C₄₄H₇₉NO₄Si₂Na (MNa)⁺764.5440, found 764.5469.

The protected vitamin 14 (0.021 g, 0.028 mmol) was dissolved in THF (2mL) and CH₃CN (2 mL). A solution of aq. 48% HF in CH₃CN (1:9 ratio, 2mL) was added at 0° C. and the resulting mixture was stirred at r.t. for8 h. Saturated aq. NaHCO₃ solution was added and the reaction mixturewas extracted with ethyl acetate. The combined organic phases werewashed with brine, dried (MgSO₄) and concentrated under reducedpressure. The residue was chromatographed on silica gel EtOAc/Hexane(80:20) to give the desired final product 15 (0.008 g, 0.019 mmol, 69%yield). The vitamin 15 was further purified by HPLC [9.4×250 mm ZorbaxSil column, 4 mL/min, Hexane/2-propanol (80:20) solvent system, Rt=8.50min).

¹H NMR (CDCl₃, 400 MHz) 6.35 (1H, d, J=11.2 Hz), 5.88 (1H, d, J=11.2Hz,), 5.11 (1H, s,), 5.09 (1H, s,), 4.62 (2H, m), 2.87-2.80 (2H, m),2.70-2.55 (3H, m), 2.35-2.26 (2H, m), 2.12 (1H, m), 0.93 (3H, d, J=6.3Hz), 0.55 (3H, s), 0.42 (2H, m), 0.34 (2H, m); ¹³C NMR (CDCl₃, 100 MHz)δ 152.00, 143.39, 130.43, 124.22, 115.28, 107.69, 71.79, 70.62, 56.43,56.30, 50.19, 45.77, 40.42, 38.17, 36.03, 33.51, 30.36, 28.94, 27.66,26,59, 24.66, 23.48, 22.27, 18.81, 12.07, 6.22; exact mass calculatedfor C₂₇H₄₄NO₂(MH)⁺413,3289, found 413.3297.

Biological Activity of CPA-1

The introduction of a methylene group to the 2-position, the removal ofthe methylene substituent at carbon 10, and the replacement of themethyl groups normally located at C-26 and C-27 as well as the hydroxylgroup normally located at C-25 with an aza group had little effect onbinding to the full length recombinant rat vitamin D receptor, ascompared to 1αa,25-dihydroxyvitamin D₃. The compound CPA-1 bound withonly slightly less affinity to the receptor as compared to the standard1,25-(OH)₂D₃ (FIG. 1), i.e. merely two logs (20 times) lower affinitycompared to 1,25(OH)₂D₃. It might be expected from these results thatcompound CPA-1 would have equivalent biological activity. Surprisingly,however, compound CPA-1 is a highly selective analog with uniquebiological activity.

FIG. 5 shows that CPA-1 is quite potent, as compared to that of1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport. CPA-1 is only about 20 timesless potent than 1,25(OH)₂D₃ in promoting active calcium transportacross the gut.

FIG. 4 demonstrates that CPA-1 has relatively low bone calciummobilization activity, as compared to 1,25(OH)₂D₃. CPA-1 is less potentthan the native hormone in releasing bone calcium stores as little to noactivity is observed until 35,100 pmol/rat is administered; whereas,significant increases in serum calcium are observed at 87 pmol and at780 pmol when the native hormone is given. Thus, CPA-1 is about 400times less potent than 1,25(OH)₂D₃ in releasing bone calcium stores.

FIG. 2 illustrates that CPA-1 has the same high potency as 1,25(OH)₂D₃on HL-60 cell differentiation, making it an excellent candidate for thetreatment of a cancer, especially for the prevention or treatment ofleukemia, colon cancer, breast cancer, skin cancer and prostate cancer.

FIG. 3 illustrates that the compound CPA-1 has less transcriptionalactivity than 1α,25-dihydroxyvitamin D₃ in bone cells. In bone cells.CPA-1 is about 10 times less potent than 1,25(OH)₂D₃ in increasingtranscription of the 24-hydroxylase gene. This result, together with thecell differentiation activity of FIG. 2, suggests that CPA-1 will bevery effective in treating the above referred to cancers because it hasdirect cellular activity in causing cell differentiation, genetranscription, and in suppressing cell growth.

Experimental Methods

The compounds of the invention were prepared and studied using thefollowing methods.

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCI) with 0.1%Chaps detergent. The receptor protein and ligand concentration wasoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In Vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24 Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (Suda et al,J. Nutr. 100:1049, 1970) (0.47% Ca)+vitamins AEK for one week followedby Diet 11 (0.02% Ca)+vitamins AEK for 3 weeks. The rats were thenswitched to the same diet containing 0.47% Ca for one week followed bytwo weeks on the same diet containing 0.02% Ca. Dose administrationbegan during the last week on 0.02% calcium diet. Four consecutive ipdoses were given approximately 24 hours apart. Twenty-four hours afterthe last dose, blood was collected from the severed neck and theconcentration of serum calcium determined by atomic absorptionspectrometry as a measure of bone calcium mobilization. The first 10 cmof the intestine was also collected for intestinal calcium transportanalysis using the everted gut sac method.

Interpretation of Data

VDR binding, HL60 cell differentiation, and transcription activity.CPA-1 (K_(i)=3×10⁻⁹M) has slightly less activity than the naturalhormone 1α,25-dihydroxyvitamin D₃ (K_(i)=2×10⁻¹¹M) in its ability tocompete with [³H]-1,25(OH)₂D₃ for binding to the full-length recombinantrat vitamin D receptor (FIG. 1). CPA-1 has the same high potency(EC₅₀=3×10⁻⁹M) in its ability (efficacy or potency) to promote HL60differentiation as compared to 1α, 25-dihydroxyvitamin D₃ (EC₅₀=2×10⁻⁹M)(See FIG. 2). Also, compound CPA-1 (EC₅₀=3×10⁻⁹M) has about 10 timesless transcriptional activity in hone cells than 1α,25-dihydroxyvitaminD₃ (EC₅₀=2×10⁻¹⁰M) (See FIG. 3). These data indicate that CPA-1 willhave significant activity as an anti-cancer agent, especially forpreventing or treating leukemia, colon cancer, breast cancer, skincancer and prostate cancer because it has direct cellular activity incausing cell differentiation and in suppressing cell growth.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of CPA-1. and 1,25(OH)₂D₃ inintestine and bone were tested. As expected, the native hormone(1,25(OH)₂D₃) increased serum calcium levels at both the 87 pmol and 780pmol dosages tested (FIG. 4). FIG. 4 also shows that CPA-1 hassignificantly less activity in mobilizing calcium from bone than1,25(OH)₂D₃. Administration of CPA-1. at 87 pmol/day, 780 pmol/day,2,340 pmol/day and 7,020 pmol/day for 4 consecutive days resulted inlittle or no mobilization of bone calcium. CPA-1 is less potent than thenative hormone in releasing bone calcium stores as little to no activityis observed until 35,100 pmol/rat is administered; whereas, significantincreases in serum calcium are observed at both 87 pmol and 780 pmolwhen the native hormone is given.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIG. 5). These results show that thecompound CPA-1 is about 20 times less potent in promoting intestinalcalcium transport activity when administered at the recommended lowerdosages, as compared to 1,25(OH)₂D₃, but its activity increases withincreasing doses in a dose dependent manner. Thus, it may be concludedthat CPA-1 has relatively high intestinal calcium transport activity atthe tested doses.

These results further illustrate that CPA-1 is an excellent candidatefor numerous human therapies as described herein. CPA-1 is an excellentcandidate for treating a cancer because: (1) it has significant VDRbinding, transcription activity and cellular differentiation activity;(2) it has low risk of hypercalcemic liability unlike 1,25(OH)₂D₃; and(3) it is easily synthesized.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I and Ia may be formulated forpharmaceutical applications as a solution in innocuous solvents, or asan emulsion, suspension or dispersion in suitable solvents or carriers,or as pills, tablets or capsules, together with solid carriers,according to conventional methods known in the art. Any suchformulations may also contain other pharmaceutically-acceptable andnon-toxic excipients such as stabilizers, anti-oxidants, binders,coloring agents or emulsifying or taste-modifying agents.

The compounds of formula I and particularly CPA-1 of formula Ia, may beadministered orally, topically, parenterally, rectally, nasally,sublingually, or transdermally. The compound is advantageouslyadministered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentarycanal, or in the form of creams, ointments, patches, or similar vehiclessuitable for transdermal applications. A dose of from 0.01 μg to 1000 μgper day of the compounds I, particularly CPA-1, preferably from about0.1 μg to about 500 μg per day, is appropriate for prevention and/ortreatment purposes, such dose being adjusted according to the disease tobe treated, its severity and the response of the subject as is wellunderstood in the art. Since the compound exhibits specificity ofaction, it may be suitably administered alone, or together with gradeddoses of another active vitamin D compound—e.g. 1α-hydroxyvitamin D₂ orD₃, or 1α,25-dihydroxyvitamin D₃—in situations where different degreesof bone mineral mobilization and calcium transport stimulation is foundto be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly CPA-1, as defined bythe above formula I and Ia as the active ingredient, and a suitablecarrier. An effective amount of such compound for use in accordance withthis invention is from about 0.01 μg to about 1000 μg per gm ofcomposition, preferably from about 0.1 μg to about 500 μg per gram ofcomposition, and may be administered topically, transdermally, orally,rectally, nasally, sublingually or parenterally in dosages of from about0.01 μg/day to about 1000 μg /day, and preferably from about 0.1 μg/dayto about 500 μg/day.

The compounds I, particularly CPA-1, may be formulated as creams,lotions, ointments, topical patches, pills, capsules or tablets,suppositories, aerosols, or in liquid form as solutions, emulsions,dispersions, or suspensions in pharmaceutically innocuous and acceptablesolvent or oils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly CPA-1, may be advantageously administeredin amounts sufficient to effect the differentiation of promyelocytes tonormal macrophages. Dosages as described above are suitable, it beingunderstood that the amounts given are to be adjusted in accordance withthe severity of the disease, and the condition and response of thesubject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from the group consisting of hydrogen and a hydroxy-protectinggroup.
 2. The compound of claim 1 wherein X₁ is hydrogen.
 3. Thecompound of claim 1 wherein X₁ and X₂ are both t-butyldimethylsilyl. 4.The compound of claim 1 wherein X₃ is t-butyloxycarbonyl.
 5. Apharmaceutical composition containing an effective amount of at leastone compound as claimed in claim 1 together with a pharmaceuticallyacceptable excipient.
 6. The pharmaceutical composition of claim 5wherein said effective amount comprises from about 0.01 μg to about 1000μg per grain of composition.
 7. The pharmaceutical composition of claim5 wherein said effective amount comprises from about 0.1 μg to about 500μg per gram of composition.
 8. A compound having the formula:

and namedN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃.
 9. A pharmaceutical composition containing an effective amount ofN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃ together with a pharmaceutically acceptable excipient.
 10. Thepharmaceutical composition of claim 9 wherein said effective amountcomprises from about 0.01 μg to about 1000 μg per gram of composition.11. The pharmaceutical composition of claim 9 wherein said effectiveamount comprise from about 0.1 μg to about 500 μg per gram ofcomposition.
 12. A method of treating a disease selected from the groupconsisting of leukemia, colon cancer, breast cancer, skin cancer orprostate cancer comprising administering to a subject with said diseasean effective amount of aN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-vitamin D analoghaving the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from the group consisting of hydrogen and a hydroxy-protectinggroup.
 13. The method of claim 12 wherein the vitamin D analog isadministered orally.
 14. The method of claim 12 wherein the vitamin Danalog is administered parenterally.
 15. The method of claim 12 whereinthe vitamin D analog is administered transdermally.
 16. The method ofclaim 12 wherein the vitamin D analog is administered rectally.
 17. Themethod of claim 12 wherein the vitamin D analog is administered nasally.18. The method of claim 12 wherein the vitamin D analog is administeredsublingually.
 19. The method of claim 12 wherein the vitamin D analog isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.20. The method of claim 12 wherein the vitamin D analog has the formula:

and namedN-cyclopropyl-(20R)-2-methylene-19,26,27-trinor-25-aza-1α-hydroxyvitaminD₃.
 21. A compound having the formula:

where X₃ is selected from the group consisting of hydrogen and ahydroxy-protecting group.
 22. The compound of claim 21 wherein X₃ ishydrogen.
 23. The compound of claim 21 wherein X₃ istert-butyloxycarbonyl.
 24. A compound having the formula:

where X₃ and X₄ , which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group.25. The compound of claim 24 wherein X₃ is hydrogen.
 26. The compound ofclaim 24 wherein X₄ is hydrogen.
 27. The compound of claim 24 wherein X₃is hydrogen and X₄ is a triethylsilyl group.
 28. The compound of claim24 wherein X₄ is hydrogen and X₃ is tert-butyloxycarbonyl.
 29. Thecompound of claim 24 wherein X₃ is tertbutyloxycarbonyl, and X₄ is atriethylsilyl group.